Fig. 4.

Ancestral and Concertina-specific substitutions of Gα₁₂ residues involved in SRE activation and Hsp90 binding. (A) An evolutionary profile of selected class-distinctive G12/13 residues is shown, with position in the primary amino acid sequence shown in superscript for Gα₁₂ and Gα₁₃ (Temple et al., 2010). (B) The indicated substitutions in myc-Gα₁₂^QL (x-axis) were examined for SRE-luciferase activation as described in Materials and Methods, and results are presented as a percent of positive control myc-Gα₁₂^QL (12QL). Panels beneath this graph show expression levels of the indicated mutants, matched with one of several different samples of myc-Gα₁₂^QL, as determined by immunoblot analysis of HEK293 cell lysates using anti-Gα₁₂ antibody. (C) Effects of the indicated substitutions within Gα₁₂ on coprecipitation by immobilized LARG and Hsp90 are shown, with lysates from cells transfected with myc-Gα₁₂^QL and empty vector analyzed in parallel. Panel at left shows blot images representative of three independent experiments, and panel at right shows precipitate-to-load ratios as a percent of the same ratio determined for myc-Gα₁₂^QL (set at 100%). Quantitative data are presented as mean ± S.E.M.